Nonalcoholic steatohepatitis (NASH) is a severe liver disorder characterized by triglyceride accumulation, severe inflammation, and fibrosis. With the recent increase in prevalence, NASH is now the leading cause of liver transplantation, with no approved therapeutics available. Despite years of research, the exact molecular mechanism of NASH progression is not well understood, but fat accumulation is believed to be the primary driver of the disease. Therefore, diacylglycerol O-acyltransferase 2 (DGAT2), a key enzyme in triglyceride synthesis, has been explored as a NASH target. RNAi-based therapeutics is revolutionizing the treatment of liver diseases, with recent chemical advances supporting long term gene silencing with single subcutaneous administration. Here we identified a hyper-functional, fully chemically stabilized GalNAc conjugated siRNA targeting DGAT2 (Dgat2-1473) that upon injection elicits up to three months of DGAT2 silencing (>80-90%, p<0.0001) in wild-type and NSG-PiZ “humanized” mice. Using an obesity-driven mouse model of NASH (ob/ob-GAN), Dgat2-1473 administration prevents and reverses triglyceride accumulation (> 50%, p:0.0008), resulting in significant improvement of the fatty liver phenotype. However, surprisingly, the reduction in liver fat didn’t translate into a similar impact on inflammation and fibrosis. Thus, while Dgat2-1473 is a practical, long-lasting silencing agent for potential therapeutic attenuation of liver steatosis, combinatorial targeting of a second pathway may be necessary for therapeutic efficacy against NASH.

Patients with type 2 diabetes (T2D) are at risk for non-alcoholic fatty liver disease (NAFLD) and associated complications. This study evaluated the performance of international (EASL-EASD-EASO) and national (DGVS) guidelines for NAFLD risk stratification. Patients with T2D prospectively underwent ultrasound, liver stiffness measurement (LSM) and serum-based fibrosis markers. Guideline-based risk classification and referral rates for different screening approaches were compared and the diagnostic properties of simplified algorithms, genetic markers and a new NASH surrogate (FAST score) were evaluated. NAFLD risk was present in 184 of 204 screened patients (age 64.2 ± 10.7 years; BMI 32.6 ± 7.6 kg/m2). EASL-EASD-EASO recommended specialist referral for 60–77% depending on the fibrosis score used, only 6% were classified as low risk. The DGVS algorithm required LSM for 76%; 25% were referred for specialised care. The sensitivities of the diagnostic pathways were 47–96%. A simplified referral strategy revealed a sensitivity/specificity of 46/88% for fibrosis risk. Application of the FAST score reduced the referral rate to 35%. This study (a) underlines the high prevalence of fibrosis risk in T2D, (b) demonstrates very high referral rates for in-depth hepatological work-up, and (c) indicates that simpler referral algorithms may produce comparably good results and could facilitate NAFLD screening.

No description available.

Patient-reported outcomes are important in nonalcoholic fatty liver disease (NAFLD). Pruritus is of special interest for evolving therapies with farnesoid X receptor (FXR) agonists. The aim of this study was to investigate the prevalence of pruritus in a real-life NAFLD cohort and analyze associations with anxiety and depression. Pruritus was assessed using a visual analogue- (VAS) and 5-D itch-scale (5-D). Anxiety and depression were evaluated by Beck’s-Depression-Inventory (BDI) and the Hospital Anxiety and Depression Scale (HADS-A, HADS-D). An optimal logistic regression model was found with a stepwise procedure to investigate variables associated with pruritus. In total, 123 NAFLD patients were recruited. VAS and 5-D were highly correlated (Spearman’s correlation coefficient 0.89). Moderate/severe pruritus was reported in 19% (VAS) and 21% (5-D) of patients. Anxiety and depression were present in 12% and 4% (HADS-A and HADS-D, respectively) and 12% (BDI) of cases. There was a significant association between VAS and BDI (p = 0.019). The final multivariate model for 5-D included diabetes mellitus (OR 4.51; p = 0.01), BDI (OR 5.98; p = 0.024), and HADS-A (OR 7.75; p = 0.011). One-fifth of NAFLD patients reported moderate or severe pruritus. 5-D was significantly associated with diabetes mellitus, depression, and anxiety. These findings should be tested in larger populations and considered in candidates for treatment with FXR agonists.

Mesenchymal stromal cell (MSC) transplantation ameliorated hepatic lipid load; tissue inflammation; and fibrosis in rodent animal models of non-alcoholic steatohepatitis (NASH) by as yet largely unknown mechanism(s). In a mouse model of NASH; we transplanted bone marrow-derived MSCs into the livers; which were analyzed one week thereafter. Combined metabolomic and proteomic data were applied to weighted gene correlation network analysis (WGCNA) and subsequent identification of key drivers. Livers were analyzed histologically and biochemically. The mechanisms of MSC action on hepatocyte lipid accumulation were studied in co-cultures of hepatocytes and MSCs by quantitative image analysis and immunocytochemistry. WGCNA and key driver analysis revealed that NASH caused the impairment of central carbon; amino acid; and lipid metabolism associated with mitochondrial and peroxisomal dysfunction; which was reversed by MSC treatment. MSC improved hepatic lipid metabolism and tissue homeostasis. In co-cultures of hepatocytes and MSCs; the decrease of lipid load was associated with the transfer of mitochondria from the MSCs to the hepatocytes via tunneling nanotubes (TNTs). Hence; MSCs may ameliorate lipid load and tissue perturbance by the donation of mitochondria to the hepatocytes. Thereby; they may provide oxidative capacity for lipid breakdown and thus promote recovery from NASH-induced metabolic impairment and tissue injury.

La stéatose hépatique non alcoolique (NAFLD) est une manifestation du syndrome métabolique dont la prévalence est en constante évolution. Les stratégies thérapeutiques sont soit difficiles à mettre en œuvre soit d’une efficacité limitée. Nous avons étudié une approche nutritionnelle avec 3 acides aminés particuliers : la glutamine, l’arginine et la citrulline (Cit) pour leurs propriétés de pharmaconutriments azotés. Dans un modèle de NAFLD modérée induite par le fructose, seule la citrulline (1 g/kg/j) permettait une amélioration du métabolisme lipidique. Toutefois, l’étude de la cinétique de NAFLD suggérait un effet protecteur du simple apport azoté. L’effet spécifique de la Cit par rapport au simple apport azoté (AANE) a donc été déterminé dans un modèle de NAFLD induite par 8 semaines de régime enrichi en fructose. Ceci a permis de confirmer l’effet protecteur de la Cit et des AANE. Toutefois, la Cit exerce un effet plus spécifique sur l’expression de Srebp1c et de Fas et améliore la disponibilité périphérique en Arg, un élément important de l’insulino-sensibilité. La stéatose est associée à une perte de masse maigre, suggérant une oxydation des AA aux dépens de l’anabolisme musculaire, et une accumulation de lipides à l’origine de la stéatose et du gain de masse grasse viscérale ; la Cit et les AANE en agissant sur la NAFLD préviendraient cet effet du fructose. Nous avons ensuite évalué les effets de la Cit dans un modèle de stéatose plus sévère induite par le western diet. La Cit améliore la fonction hépatique (diminution des lipides et de l’inflammation hépatique) et préserve l’axe intestin-foie (restauration du groupe Bacteroides/Prevotella dans la muqueuse colique, diminution de l’inflammation intestinale et augmentation de l’expression de la Claudine 1) mais ne permet pas de prévenir l’ensemble des altérations liées au western diet. Il serait intéressant d’évaluer la relation effet/dose et l’efficacité de la Cit en association avec d’autres traitements. Par ailleurs, les mécanismes cellulaires restent à élucider. / Nonalcoholic fatty liver disease (NAFLD) is a manifestation of the metabolic syndrome whose prevalence is constantly growing. Therapeutic strategies are either difficult to implement or of limited effectiveness. We studied a nutritional approach with three specific amino acids: glutamine, arginine and citrulline (Cit) for their pharmaconutrient properties. In a model of moderate fructose-induced NAFLD, citrulline alone (1 g/kg/day) improved lipid metabolism. However, the study of the kinetics of NAFLD suggested a protective effect of nitrogen supply by itself. The specific effect of Cit compared to that of nitrogen (NEAAs) has been determined in a model of 8 week fructose diet-induced NAFLD. This has confirmed the protective effect of Cit and NEAAs. However, Cit exerted a specific effect on the expression of Fas and SREBP1c and improves peripheral Arg availability, an important component of insulin sensitivity. Steatosis was associated with loss of lean mass, suggesting AA oxidation at the expense of muscle anabolism, and lipid accumulation causing steatosis and visceral fat gain; Cit and NEAAs by acting on NAFLD would prevent this effect of fructose. We then evaluated the effects of Cit in a model of more severe steatosis induced by western diet. Cit improved liver function (reduced fat and liver inflammation) and protected the liver-gut axis (restoration of Bacteroides/Prevotella group in the colonic mucosa, decreased intestinal inflammation and increased expression of claudin 1) but did not prevent all western diet-induced alterations. It would be interesting to assess the dose/effect relationship and the effectiveness of Cit in combination with other treatments. Furthermore, the cellular mechanisms remain to be elucidated.

A doença hepática gordurosa não alcoólica (DHGNA) abrange alterações desde esteatose até esteato-hepatite não alcoólica (EHNA), podendo evoluir para fibrose, cirrose e carcinoma hepatocelular. A DHGNA é considerada a doença hepática mais comum na atualidade e com prevalência mundial alarmante. Esta doença caracteriza-se, basicamente, pela deposição de triglicérides nos hepatócitos, podendo evoluir com inflamação e fibrose, e está intimamente associada com resistência à insulina (RI), diabetes mellitus tipo 2 e obesidade. Os hepatócitos representam as principais células hepáticas e se comunicam através de junções do tipo gap, formadas principalmente por conexina 32 (Cx32). Esta proteína apresenta importante função no controle da homeostase tecidual, regulando processos fisiológicos e tem sido associada como agente protetor na hepatocarcinogênese e outros processos patológicos, porem pouco se sabe sobre sua participação na DHGNA. Sendo assim, o objetivo deste trabalho foi avaliar a participação da Cx32 na fisiopatogênese da DHGNA, utilizando camundongos knockout para Cx32 (Cx32-KO) submetidos a uma dieta hiperlipídica deficiente em colina. Foram analisados dados biométricos, histopatológicos, função hepática, RI, citocinas inflamatórias, adipocinas, estresse oxidativo, peroxidação lipídica e a expressão de genes envolvidos na DHGNA. Os animais Cx32-KO apresentaram maior acumulo de triglicérides hepáticos em relação aos animais selvagens e, consequentemente, maior peso absoluto e relativo do fígado. Adicionalmente, apresentaram maior inflamação hepática demonstrado pela exacerbação da citocina TNF-&#945; e supressão da IL-10, maior dano hepatocelular indicado pelo aumento das enzimas AST e ALT, aumento da peroxidação lipídica e alterações na expressão de genes chaves na fisiopatogênese da DHGNA, como SREBP1c. No entanto, não houve diferença nos marcadores histopatológicos, RI e estresse oxidativo hepático. Por fim, os animais Cx32-KO apresentaram maior produção de leptina e adiponectina no tecido adiposo. Todos esses resultados revelam que a Cx32 pode atuar como um agente protetor ao desenvolvimento da DHGNA, sugerindo seu potencial como novo alvo terapêutico / Nonalcoholic fatty liver disease (NAFLD) covers a spectrum of liver diseases ranging from steatosis to nonalcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis and eventually hepatocellular carcinoma. NAFLD is currently the most common liver disease in the world with an alarming prevalence worldwide. This disease is characterized by the deposition of triglycerides in hepatocytes and can develop inflammation and fibrosis. NAFLD is closely associated with insulin resistance (IR), type 2 diabetes mellitus and obesity. The gap junctions mediate intercellular communication and are critical for maintaining integral cellular processes. In hepatocytes, the major type of liver cells, the gap junctions are formed mainly by connexin 32 (Cx32). This protein is important for the control of tissue homeostasis and for physiological processesregulation. It has been linked as a protective agent in hepatocarcinogenesis and other pathological processes. However, little is known about its role on NAFLD. Thus, the aim of this study was to evaluate the participation of Cx32 in the pathogenesis of NAFLD using WT and Cx32-knockout mice (Cx32-KO) subjected to a high-fat diet deficient in choline. NAFLD was evaluated based on a battery of clinically relevant read-outs, including histopathological examination, inflammatory citokines, liver damage markers, in-depth lipid analysis, assessment of oxidative stress, insulin and glucose tolerance and lipid-related biomarkers.The Cx32-KO animals showed higher accumulation of hepatic triglycerides, increased inflammation and lipid peroxidation and increased production of leptin and adiponectin in the adipose tissue when compared with WT. Moreover, there was no difference in histopathological markers, RI and hepatic oxidative stress. Taken together, all these results show that Cx32 is a protective agent to the development of the disease, suggesting its potential as a novel therapeutic target in NAFLD

No description available.

A doença hepática gordurosa não alcoólica (DHGNA) abrange alterações desde esteatose até esteato-hepatite não alcoólica (EHNA), podendo evoluir para fibrose, cirrose e carcinoma hepatocelular. A DHGNA é considerada a doença hepática mais comum na atualidade e com prevalência mundial alarmante. Esta doença caracteriza-se, basicamente, pela deposição de triglicérides nos hepatócitos, podendo evoluir com inflamação e fibrose, e está intimamente associada com resistência à insulina (RI), diabetes mellitus tipo 2 e obesidade. Os hepatócitos representam as principais células hepáticas e se comunicam através de junções do tipo gap, formadas principalmente por conexina 32 (Cx32). Esta proteína apresenta importante função no controle da homeostase tecidual, regulando processos fisiológicos e tem sido associada como agente protetor na hepatocarcinogênese e outros processos patológicos, porem pouco se sabe sobre sua participação na DHGNA. Sendo assim, o objetivo deste trabalho foi avaliar a participação da Cx32 na fisiopatogênese da DHGNA, utilizando camundongos knockout para Cx32 (Cx32-KO) submetidos a uma dieta hiperlipídica deficiente em colina. Foram analisados dados biométricos, histopatológicos, função hepática, RI, citocinas inflamatórias, adipocinas, estresse oxidativo, peroxidação lipídica e a expressão de genes envolvidos na DHGNA. Os animais Cx32-KO apresentaram maior acumulo de triglicérides hepáticos em relação aos animais selvagens e, consequentemente, maior peso absoluto e relativo do fígado. Adicionalmente, apresentaram maior inflamação hepática demonstrado pela exacerbação da citocina TNF-&#945; e supressão da IL-10, maior dano hepatocelular indicado pelo aumento das enzimas AST e ALT, aumento da peroxidação lipídica e alterações na expressão de genes chaves na fisiopatogênese da DHGNA, como SREBP1c. No entanto, não houve diferença nos marcadores histopatológicos, RI e estresse oxidativo hepático. Por fim, os animais Cx32-KO apresentaram maior produção de leptina e adiponectina no tecido adiposo. Todos esses resultados revelam que a Cx32 pode atuar como um agente protetor ao desenvolvimento da DHGNA, sugerindo seu potencial como novo alvo terapêutico / Nonalcoholic fatty liver disease (NAFLD) covers a spectrum of liver diseases ranging from steatosis to nonalcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis and eventually hepatocellular carcinoma. NAFLD is currently the most common liver disease in the world with an alarming prevalence worldwide. This disease is characterized by the deposition of triglycerides in hepatocytes and can develop inflammation and fibrosis. NAFLD is closely associated with insulin resistance (IR), type 2 diabetes mellitus and obesity. The gap junctions mediate intercellular communication and are critical for maintaining integral cellular processes. In hepatocytes, the major type of liver cells, the gap junctions are formed mainly by connexin 32 (Cx32). This protein is important for the control of tissue homeostasis and for physiological processesregulation. It has been linked as a protective agent in hepatocarcinogenesis and other pathological processes. However, little is known about its role on NAFLD. Thus, the aim of this study was to evaluate the participation of Cx32 in the pathogenesis of NAFLD using WT and Cx32-knockout mice (Cx32-KO) subjected to a high-fat diet deficient in choline. NAFLD was evaluated based on a battery of clinically relevant read-outs, including histopathological examination, inflammatory citokines, liver damage markers, in-depth lipid analysis, assessment of oxidative stress, insulin and glucose tolerance and lipid-related biomarkers.The Cx32-KO animals showed higher accumulation of hepatic triglycerides, increased inflammation and lipid peroxidation and increased production of leptin and adiponectin in the adipose tissue when compared with WT. Moreover, there was no difference in histopathological markers, RI and hepatic oxidative stress. Taken together, all these results show that Cx32 is a protective agent to the development of the disease, suggesting its potential as a novel therapeutic target in NAFLD

Made available in DSpace on 2015-07-22T20:50:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-05-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Objetivo: Avaliar o efeito da intervenção nutricional como tratamento exclusivo na resistência à insulina, parâmetros bioquímicos de síndrome metabólica e grau de esteatose hepática em portadores de doença hepática gordurosa não alcoólica (DHGNA). Métodos: 31 portadores de DHGNA, diagnosticados por tomografia computadorizada e/ou biópsia hepática receberam dieta com restrição de 500 a 1000 kcal/dia. O valor energético total (VET) foi distribuído em 15% proteína, 55% carboidrato e 30% gordura. Foram avaliados no início do estudo e após 6 meses de tratamento: grau de esteatose hepática e obesidade visceral por meio de tomografia computadorizada. Níveis séricos de alanina aminotransferase (ALT), gama glutamil transferase (GGT), aspartato aminotransferase (AST), e fosfatase alcalina, e parâmetros de síndrome metabólica (SM), glicemia, triglicerídeos e da lipoproteína de alta intensidade (HDL-C) foram medidos por método automatizado. As medidas antropométricas, índice de massa corpórea (IMC), circunferência da cintura (CC) e relação cintura/quadril e o consumo alimentar (pelo registro alimentar de 7 dias) foram avaliados mensalmente. Ao final do acompanhamento, os pacientes foram considerados como aderentes ou não aderentes de acordo com perda de peso maior ou menor que 5% do peso inicial, respectivamente. Os testes de Mann-Whitney, quiquadrado e Wilcoxon foram utilizados na análise estatística. Resultados: Dos 31 pacientes incluídos, 17 foram classificados como aderentes (grupo 1) e 14 como não aderentes (grupo 2). No grupo 2 foi observada redução significante dos valores do índice de massa corpórea (IMC), circunferência da cintura (CC) , enquanto entre os pacientes que aderiram à dieta, além da melhora significante de todos os parâmetros antropométricos, também houve redução estatisticamente significante nos níveis da alanina aminotransferase (ALT) , gama glutamil transferase (GGT), insulina, HOMA–IR, gordura visceral, gordura total, e da densidade tomográfica do fígado, assim como aumento da lipoproteína de alta intensidade (HDL-C). Nesses pacientes houve diminuição estatisticamente significante do valor calórico total com diminuição do consumo de gordura total e saturada. Conclusão: O tratamento nutricional como terapia exclusiva, com a perda de pelo menos 5% de peso inicial, foi capaz de modificar os parâmetros metabólicos de síndrome metabólica, valores das enzimas hepáticas e o grau de esteatose em portadores de doença hepática gordurosa não alcoólica (DHGNA), demonstrando que a intervenção dietética é efetiva no tratamento da doença. / Purpose: Evaluate the effect of diet therapy as exclusive treatment on insulin resistance, biochemical parameters of metabolic syndrome and degree of hepatic steatosis in patients with nonalcoolic fatty liver disease (NAFLD). Methods: Thirty-one patients diagnosed with NAFLD by computed tomography and/or liver biopsy received a restricted diet of energy (reduction of 500 to 100 kcal/day) containing 15% protein, 55% carbohydrates and 30% fat. The following parameters were evaluated at the entry and 6 months after dietary instructions: the degree of hepatic steatosis and visceral obesity by computed tomography, the serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT) levels, metabolic syndrome parameters, glycemia, triglycerides and high density liprotein (HDL-c) were measured by an automated method. Insulin concentration was determined by immunofluorometry and insulin resistance (IR) was calculated by homeostasis model assessment (HOMA-IR). Anthropometric variables, body mass index (BMI), waist circumference, waist-to-hip ratio, and food intake (7-day diary) were evaluated monthly. At the end of follow-up, the patients were classified as adherent or non-adherent to treatment according to a weight loss of more or less than 5% of initial body weight, respectively. Results were analyzed statistically using the Mann- Whitney, chi-square and Wilcoxon tests. Results: Seventeen of the 31 patients were classified as adherent (group 1) and 14 as non-adherent (group 2). Group 2 presented only a significant reduction in body mass index (BMI) and waist circumference. In contrast, in group 1, in addition to significant improvement of all anthropometric parameters, a significant reduction was observed in alanine aminotransferase (ALT) and gamma-glutamyl transferase (GGT) levels, homeostasis model assessment (HOMA-IR), visceral fat and tomographic liver density, together with an increase in high density liprotein (HDL-c) serum levels. These patients decrease presented a significant in total energy intake and in the amount of total and saturated fat. Conclusions: Nutritional intervention as exclusive treatment, with a loss of at least 5% of initial weight, was able to modify metabolic syndrome parameters, liver enzymes and degree of steatosis in patients with NAFLD, demonstrating that dietary intervention is effective in the treatment of this disease. / TEDE / BV UNIFESP: Teses e dissertações